Alternative mRNA isoforms are an important means of

Alternative mRNA isoforms are an important means of diversifying protein behavior, and have contributed to the complexity of vertebrate organisms (Roy et al., 2013). Here we show that two Dido isoforms generated by differential splicing are critical to determination of cell lineage early in ESC differentiation. Self-renewal and differentiation events apparently have distinct genetic requirements in the transcription modes for pluripotent ESCs and for differentiating cells, which must integrate all signals needed for each stage. DIDO1 and DIDO3 have several mechanisms that might take part in the pluripotent and somatic cell transcription modes and in development. The composition of DIDO isoform domains might aid in establishing these modes. DIDO1 has the PHD, whereas DIDO3 encodes a much larger protein with three signature motifs, PHD, TFS2M, and SPOC, all typically found in proteins involved in transcription regulation. We found that DIDO3 binds RNA POL II and thus links DIDO to transcriptional control, as shown for the yeast DIDO homolog BYE1 (Kinkelin et al., 2013; Pinskaya et al., 2014). We show a direct DIDO3 role in regulating its own expression and that of DIDO1, as the Dido3ΔCT loss-of-function mutation confers a cell phenotype similar to that seen when DIDO1 is misregulated. ChIP mapping of DIDO3 recruitment to the Dido locus also indicated a binding pattern associated with a region that includes, but is not limited to, the Dido promoter (Figure 7). Results from Go 6983 binding indicated that DIDO3 binding to its own genomic DNA modulates Dido1 expression and might participate in regulating core stemness genes, a question currently under study. These findings coincide with the role of d(PPS), the DIDO homolog in Drosophila. d(PPS) protein also has four signature motifs typically found in proteins that act in transcriptional regulation; d(PPS) is needed to regulate Sxl (sex-lethal) splicing (Johnson et al., 2010) and DIDO3 is necessary for DIDO1 expression. Given the known d(PPS) splicing function, our data and the parallels with d(PPS) results suggest a role for DIDO in transcription control by splicing.
Experimental Procedures
Author Contributions
Acknowledgments We thank the CNB Proteomics facility for peptide synthesis, the CNB Genomic Service Unit for performing microarray experiments, and C. Mark for editorial assistance. This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (MINECO-FEDER SAF2013-42289-R and SAF2016-75456-R) and the Alfonso Martín Escudero Foundation.
Introduction Sickle cell anemia, one of humankind\'s most common hereditary monogenic diseases, is an emerging global health burden. In the United States, approximately 100,000 people are affected, annual mortality approaches 4%, and the costs of medical care exceed $1.1 billion (Kauf et al., 2009). Moreover, sickle cell disease is designated by the World Health Organization as a public health priority, with 300,000 births yearly, and it is estimated that 10 million African, Arab, and Indian individuals will be living with this disease in the future (Piel et al., 2013a, 2013b). In underdeveloped countries, this is a disease of childhood where most of the affected die young. With access to high-quality medical care, survival into the seventh and eighth decades is possible. Hydroxyurea is the sole approved drug treatment that alters disease pathophysiology by increasing the level of fetal hemoglobin (HbF). HbF has the property of inhibiting the polymerization of deoxy sickle hemoglobin (HbS), which is the proximal driver of disease pathophysiology (Steinberg et al., 2009). As part of a large NIH-funded NextGen Consortium, we generated a comprehensive library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities, β-globin gene (HBB) haplotypes, and HbF levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Access to a genetically diverse cohort of sickle-cell-disease-specific iPSCs provides a unique resource for the study of novel haplotype-specific polymorphisms that affect disease severity as well as the development of novel patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell and gene-based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation.